Circadian clock rhythms in different adipose tissue model systems

ABSTRACT

Circadian clock-controlled 24-h oscillations in adipose tissues play an important role in the regulation of energy homeostasis, thus representing a potential drug target for prevention and therapy of metabolic diseases. For pharmacological screens, scalable adipose model systems are needed that largely recapitulate clock properties observed in vivo. In this study, we compared molecular circadian clock regulation in different ex vivo and in vitro models derived from murine adipose tissues. Explant cultures from three different adipose depots of PER2::LUC circadian reporter mice revealed stable and comparable rhythms of luminescence ex vivo. Likewise, primary pre- and mature adipocytes from these mice displayed stable luminescence rhythms, but with strong damping in mature adipocytes. Stable circadian periods were also observed using Bmal1-luc and Per2-luc reporters after lentiviral transduction of wild-type pre-adipocytes. SV40 immortalized adipocytes of murine brown, subcutaneous and epididymal adipose tissue origin showed rhythmic mRNA expression of the core clock genes Bmal1, Per2, Dbp and REV-erbα in pre- and mature adipocytes, with a maturation-associated increase in overall mRNA levels and amplitudes. A comparison of clock gene mRNA rhythm phases revealed specific changes between in vivo and ex vivo conditions. In summary, our data indicate that adipose culture systems to a large extent mimic in vivo tissue clock regulation. Thus, both explant and cell systems may be useful tools for large-scale screens for adipose clock regulating factors.     

Circadian clock gene expression regulates cancer cell growth through glutaminase

Glutamine is an essential amino acid for malignant tumor cells. Glutaminase that metabolizes glutamine reaches a maximum expression in tumors immediately before the maximum proliferation rate. Tumor cells grow at different rates during the day. We postulated that the activity of glutaminase in tumor cells is subject to the regulation of circadian clock gene. We measured glutaminase by western blot analysis and circadian clock gene expression by real-time polymerase chain reaction in the liver and tumor cells at six equispaced time points of the day in individual mice of a 12/12 h light/dark schedule. The results showed that the tumor-bearing mice, under normal diurnal conditions, are circadianly entrained, as reflected by the normal host locomotor activity rhythms and rhythmic liver clock gene expression. The tumors within these mice are also circadianly organized, as reflected by circadian clock gene （Bmall） expression. What is most remarkable is that kidney-type glutaminase also showed circadian rhythms in the same pattern with tumor circadian clock gene expression in liver cancer xenograft model, indicating that conditionally inhibiting glutaminase activity may provide a new target for cancer therapy.     

昆虫钟基因研究进展

昆虫进化形成了内在的生物钟机制以协调行为、生理及代谢节律与外部环境信号同步,从而更有效地利用资源并获得适应性优势。行为、生理及代谢昼夜调控的协调对于昆虫有效应对可预见的生理上的挑战至关重要。生化过程和代谢变化与外部环境的昼夜节律同步性受基因表达的控制,钟基因在昆虫的重要生理过程如中枢及外围生物钟机制、光周期信号传导、光周期介导的外围组织调控、代谢以及免疫中发挥着重要作用。根据信号转导过程中的作用,昆虫钟基因分为3类——信号输入基因、信号震荡起搏器和信号输出基因,它们通过相互作用形成了复杂的转录-翻译反馈回路并参与调控昆虫昼夜节律和光周期事件。本文针对昆虫钟基因的鉴定、分类和功能,作用分子机制以及研究方法和挑战等方面作了总结,并展望了昆虫钟基因未来的研究方向,这将为昆虫钟基因的进一步功能研究及开发利用提供信息参考。     

Interactions of polymorphisms in different clock genes associated with circadian phenotypes in humans

Several studies have shown that mutations and polymorphisms in clock genes are associated with abnormal circadian parameters in humans and also with more subtle non-pathological phenotypes like chronotypes. However, there have been conflicting results, and none of these studies analyzed the combined effects of more than one clock gene. Up to date, association studies in humans have focused on the analysis of only one clock gene per study. Since these genes encode proteins that physically interact with each other, combinations of polymorphisms in different clock genes could have a synergistic or an inhibitory effect upon circadian phenotypes. In the present study, we analyzed the combined effects of four polymorphisms in four clock genes (Per2, Per3, Clock and Bmal1) in people with extreme diurnal preferences (morning or evening). We found that a specific combination of polymorphisms in these genes is more frequent in people who have a morning preference for activity and there is a different combination in individuals with an evening preference for activity. Taken together, these results show that it is possible to detect clock gene interactions associated with human circadian phenotypes and bring an innovative idea of building a clock gene variation map that may be applied to human circadian biology.     

Circadian clock genes promote glioma progression by affecting tumour immune infiltration and tumour cell proliferation

ObjectivesCircadian rhythm controls complicated physiological activities in organisms. Circadian clock genes have been related to tumour progression, but its role in glioma is unknown. Therefore, we explored the relationship between dysregulated circadian clock genes and glioma progression.Materials and MethodsSamples were divided into different groups based on circadian clock gene expression in training dataset (n = 672) and we verified the results in other four validating datasets (n = 1570). The GO and GSEA enrichment analysis were conducted to explore potential mechanism of how circadian clock genes affected glioma progression. The single‐cell RNA‐Seq analysis was conducted to verified previous results. The immune landscape was evaluated by the ssGSEA and CIBERSORT algorithm. Cell proliferation and viability were confirmed by the CCK8 assay, colony‐forming assay and flow cytometry.ResultsThe cluster and risk model based on circadian clock gene expression can predict survival outcome. Samples were scoring by the least absolute shrinkage and selection operator regression analysis, and high scoring tumour was associated with worse survival outcome. Samples in high‐risk group manifested higher activation of immune pathway and cell cycle. Tumour immune landscape suggested high‐risk tumour infiltrated more immunocytes and more sensitivity to immunotherapy. Interfering TIMELESS expression affected circadian clock gene expression, inhibited tumour cell proliferation and arrested cell cycle at the G0/G1 phase.ConclusionsDysregulated circadian clock gene expression can affect glioma progression by affecting tumour immune landscape and cell cycle. The risk model can predict glioma survival outcome, and this model can also be applied to pan‐cancer.     

果蝇昼夜节律的分子机制研究进展

果蝇由于遗传易操作性而成为一个研究昼夜节律分子机制的理想模式生物 . 到目前为止,通过遗传学和生物化学方法已经鉴定到 10 多个时钟基因 (clock genes) 和许多时钟相关基因,包括时钟输入基因和钟控基因 . 这些时钟基因以及它们的相应产物组成两个互相依赖的转录 / 翻译反馈环路,从而调节行为和生理的昼夜节律 . 果蝇这种核心钟的工作原理同样见于哺乳动物 .     

生物发光成像无损伤研究植物生物钟的方法

植物生物钟系统是植物为了适应地球自转进化出的以约24小时为周期的分子系统, 通过感知并整合外界周期性变化的环境信号进而协调细胞内相应基因的表达和能量状态, 赋予植物对生存环境的适应性并参与调控多个植物生长发育过程。目前, 越来越多的研究聚焦于解析植物生物钟的分子机制, 基于此也衍生出很多研究生物钟表型的方法。该文在总结已有生物钟检测方法的基础上, 重点介绍生物钟表型实验中最常用且比较稳定可靠的实验方法, 以期为生物钟的表型研究尤其是生物钟机制研究提供技术支持与借鉴。     

Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function

Plant chloroplasts are not only the main cellular location for storage of elemental iron (Fe), but also the main site for Fe, which is incorporated into chlorophyll, haem and the photosynthetic machinery. How plants measure internal Fe levels is unknown. We describe here a new Fe‐dependent response, a change in the period of the circadian clock. In Arabidopsis, the period lengthens when Fe becomes limiting, and gradually shortens as external Fe levels increase. Etiolated seedlings or light‐grown plants treated with plastid translation inhibitors do not respond to changes in Fe supply, pointing to developed chloroplasts as central hubs for circadian Fe sensing. Phytochrome‐deficient mutants maintain a short period even under Fe deficiency, stressing the role of early light signalling in coupling the clock to Fe responses. Further mutant and pharmacological analyses suggest that known players in plastid‐to‐nucleus signalling do not directly participate in Fe sensing. We propose that the sensor governing circadian Fe responses defines a new retrograde pathway that involves a plastid‐encoded protein that depends on phytochromes and the functional state of chloroplasts.     

Human intestinal circadian clock: expression of clock genes in colonocytes lining the crypt

Biological clock components have been detected in many epithelial tissues of the digestive tract of mammals (oral mucosa, pancreas, and liver), suggesting the existence of peripheral circadian clocks that may be entrainable by food. Our aim was to investigate the expression of main peripheral clock genes in colonocytes of healthy humans and in human colon carcinoma cell lines. The presence of clock components was investigated in single intact colonic crypts isolated by chelation from the biopsies of 25 patients (free of any sign of colonic lesions) undergoing routine colonoscopy and in cell lines of human colon carcinoma (Caco2 and HT29 clone 19A). Per-1, per-2, and clock mRNA were detected by real-time RT-PCR. The three-dimensional distributions of PER-1, PER-2, CLOCK, and BMAL1 proteins were recorded along colonic crypts by immunofluorescent confocal imaging. We demonstrate the presence of per-1, per-2, and clock mRNA in samples prepared from colonic crypts of 5 patients and in all cell lines. We also demonstrate the presence of two circadian clock proteins, PER-1 and CLOCK, in human colonocytes on crypts isolated from 20 patients (15 patients for PER-1 and 6 for CLOCK) and in colon carcinoma cells. Establishing the presence of clock proteins in human colonic crypts is the first step toward the study of the regulation of the intestinal circadian clock by nutrients and feeding rhythms.     

昼夜节律钟调控代谢的研究进展

生理和行为的昼夜节律性调控对健康生活是必需的。越来越多的流行病学和遗传学证据显示昼夜节律的破坏与代谢紊乱性疾病相关联。在分子水平上,昼夜节律受到时钟蛋白组成的转录一翻译负反馈环的调控。时钟蛋白通过以下两种途径调节代谢：首先,时钟蛋白作为转录因子直接调节一些代谢关键步骤的限速酶和代谢相关核受体的表达,其次作为代谢相关核受体的辅调节因子来激活或抑制其转录活性。虽然时钟蛋白对代谢途径的调节导致代谢物水平呈昼夜节律振荡,但是产生的代谢物反过来又可以影响昼夜节律钟基因的表达,进而影响昼夜节律钟。深入研究昼夜节律钟与代谢的交互调节可能为治疗某些代谢紊乱性疾病提供新的治疗方案。     

The role of circadian clock genes in the photoperiodic timer of the linden bug Pyrrhocoris apterus during the nymphal stage

Many insects survive seasonal adversities during diapause, a form of programmed developmental and metabolic arrest. Photoperiodically regulated entry into diapause allows multivoltine insect species to optimize the number of generations. The molecular mechanism of the photoperiodic timer is unknown in insects. In the present study, we take advantage of the robust reproductive diapause response in the linden bug Pyrrhocoris apterus and explore the fifth‐instar nymphal stage, which is the most photoperiod‐sensitive stage. The nymphs display daily changes in locomotor activity during short days; this differs from the activity observed during long days. We find evidence of cyclical expression of the circadian clock genes, per and cyc, in nymphal heads; in addition, per expression is also photoperiod‐dependent. The RNA interference‐mediated knockdown of the two circadian clock genes, Clk and cyc, during the nymphal stage results in reproductive arrest in adult females. Furthermore, Clk and cyc knockdown induces the expression of the storage protein hexamerin in the fat body, whereas the expression of vitellogenin diminishes. Taken together, these data support the involvement of circadian clock genes in photoperiodic timer and/or diapause induction.     

Several clock genes polymorphisms are meaningful risk factors in the development and severity of cannabis addiction

Circadian rhythms have been related to psychiatric diseases and regulation of dopaminergic transmission, especially in substance abusers. The relationship between them remained enigmatic and no data on the role of clock genes on cannabis dependence have been documented. We aimed at exploring the role of clock gene genotypes as potential predisposing factor to cannabis addiction, using a high throughput mass spectrometry methodology that enables the large-scale analysis of the known relevant polymorphisms of the clock genes. We have conducted a case-control study on 177 Caucasians categorizing between cannabis-addicted subjects and casual consumers based on structured interviews recorded socio-demographic data, AUDIT, Fagerström test, MINI, and medical examinations. Alcohol, opiates, and stimulants’ consumption was as exclusion criteria. We report an association between several Single Nucleotide Polymorphism (SNP)s in main circadian genes SNPs, especially the gene locus HES7/PER1 on chromosome 17 and cannabis consumption as well as the development of neuropsychiatric and social disorders. This SNP’s signature that may represent a meaningful risk factor in the development of cannabis dependence and its severity requires to be deeply explored in a prospective study.     

棉铃虫生物钟基因HeDbt的克隆和表达模式分析

【目的】克隆并分析棉铃虫Helicoverpa armigera生物钟基因Double-time (Dbt),明确该基因的昼夜表达模式,探讨其表达水平的影响因子,为研究夜蛾科昆虫复眼中生物钟基因的作用机制奠定基础,为理解外周组织中生物钟基因功能提供参考。【方法】采用RT-PCR和RACE技术从2日龄棉铃虫雌成虫复眼中克隆生物钟基因Dbt,并利用在线网站和软件进行生物信息学分析。采用qPCR技术检测棉铃虫雌、雄成虫不同组织(头、脑、复眼、触角、胸、腹、足和翅)中Dbt的表达水平;检测光周期14L∶10D和持续黑暗(DD)下雌、雄成虫头和复眼中Dbt的昼夜表达模式;在暗期用棉铃虫敏感波段光(UV、蓝光和绿光)照射2日龄成虫6 h,检测复眼中Dbt表达水平的变化;在暗期进行雌、雄成虫交配,检测交配结束及3 h后复眼中Dbt表达水平的变化。【结果】成功克隆到棉铃虫生物钟基因Dbt的cDNA序列,命名为HeDbt(GenBank登录号: KM233159),开放阅读框长1 026 bp,编码314个氨基酸组成的多肽。HeDbt理论推测分子量为39.79 kD,等电点(pI)为9.55,不具有跨膜拓扑结构,包含典型的昆虫DBT蛋白保守区域,其与甜菜夜蛾Spodoptera exigua和柞蚕Antheraea pernyi DBT的同源性较高, 氨基酸序列一致性分别为99%和97%。qPCR结果表明,HeDbt在成虫各组织中均有表达,在头、脑和复眼中表达水平较低,在胸和腹中表达水平较高;在14L∶10D和DD下,头和复眼中HeDbt未呈现明显的昼夜表达节律。暗期光照和交配后,复眼中HeDbt的表达均显著下调,但雌、雄成虫间HeDbt表达水平整体相似。【结论】成功克隆得到棉铃虫生物钟基因HeDbt,其在棉铃虫成虫头和复眼中表达水平较低,且不具有昼夜规律性,但复眼中Dbt的表达受到光照和交配的影响。本研究为进一步探索夜蛾外周组织生物钟基因功能奠定了基础。     

Effects of iron status on expression of circadian clock genes and serum lipid metabolism in sucking piglets

The aim of the study is to determine the effects of iron on circadian clock gene expression and serum lipid metabolism in sucking piglets. Twenty-four neonatal piglets were selected and randomly assigned into three groups (A, B, and C) with eight replicates. Group A were received 1 mL physiological saline by intramuscular administration at d 3 and d 10; group B were received 1 mL iron dextran (100 mg) by intramuscular administration at d 3 and 1 mL physiological saline at d 10, respectively; group C were received 1 mL of iron dextran (100 mg) by intramuscular administration at both d 3 and d 10. Our results reveal that the relative expressions of Cry1, Cry2, Per1, Per2, and Bmal in liver were significantly different in the three groups (p < 0.05). Meanwhile, the content of triglyceride (TG) and high-density lipoprotein (HDL) in serum were also affected by the iron supplementation (p < 0.05). These results indicated that iron affected hepatic circadian clock genes significantly, meanwhile, it may possible association with lipid metabolism.     

生物钟在卵巢生理和病理中的作用

哺乳动物的昼夜节律是基因编码的分子钟在体内产生的一种以大约24 h为周期的生理现象,使机体的生理过程与外界环境的变化相协调,是对环境适应的一种表现.在哺乳动物中,繁殖生理功能受生物钟系统的调节.在下丘脑-垂体-卵巢(hypothalamic-pituitary-ovarian,HPO)轴的各组织中均已观察到生物钟基因的...     

The Tilapia collagen peptide mixture TY001 protects against LPS-induced inflammation,disruption of glucose metabolism,and aberrant expression of circadian clock genes in mice

The Tilapia collagen peptide mixture TY001 has been shown to accelerate wound healing in streptozotocin-induced diabetic mice and to protect against streptozotocin-induced inflammation and elevation in blood glucose. The goals of the present study are to further study TY001 effects on lipopolysaccharide (LPS)-induced inflammation and metabolic syndrome. LPS is known to disrupt circadian clock to produce toxic effects, the effects of TY001 on rhythmic alterations of serum cytokines and hepatic clock gene expressions were examined. Mice were given TY001 (30 g/L, ≈ 40 g/kg) through the drinking water for 30 days, and on the 21^st day of TY001 supplementation, LPS (0.25 mg/kg, ip, daily) was given for 9 days to establish the inflammation model. Repeated LPS injections produced inflammation, impaired glucose metabolism, and suppressed the expression of circadian clock core genes Bmal1 and Clock; clock feedback gene Cry1, Cry2, Per1, and Per2; clock target gene Rev-erbα and RORα. TY001 prevented LPS-induced elevations of TNFα, IL-1β, IL-6, and IL-10 in the liver, along with improved histopathology. TY001 reduced LPS-elevated fasting blood glucose and increased LPS-reduced serum insulin levels, probably via increased glucose transporter GLUT2, enhanced insulin signaling p-Akt and p-IRS-1^Try612. Importantly, LPS-induced circadian elevations of serum TNFα and IL-1β and aberrant expression of circadian clock genes in the liver were ameliorated by TY001. Immunohistochemistry revealed that the LPS decreased Bmal1 and Clock protein in the liver, which was recovered by TY001. Taken together, TY001 is effective against LPS-induced inflammation, disruption of glucose metabolism and disruption of circadian clock gene expressions.

Abbreviations: TY001: Tilapia collagen peptide mixture; LPS: Lipopolysaccharide; TNFα: Tumor necrosis factor-α; IL-1β: Interleukin-1β; GLUT2: Glucose transporter 2     

蓝藻生物钟分子机理的研究进展

蓝藻是具有内源性生物钟的简单生物.虽然蓝藻生物钟具有跟真核生物同样的基础特征,但其相关基因和蛋白质与真核生物没有同源性.蓝藻生物钟的核心是kai基因簇及其编码的蛋白KaiA,KaiB和KaiC.这三种Kai蛋白相互作用调节KaiC的磷酸化状态,从而产生昼夜节律信息.KaiC的磷酸化循环是昼夜节律的起博器,调控包括kai基因在内的相关基因的节律性表达.组氨酸蛋白激酶的磷酸化传递可将环境信息输入和将节律信息输出生物钟核心.